Comparative inhibition of rabbit globin mRNA translation by modified antisense oligodeoxynucleotides

Abstract

We have studied the translation of rabbit globin mRNA in cell free systems (reticulocyte lysate and wheat germ extract) and in microinjected Xenopus oocytes in the presence of anti-sense oligodeoxynucleotides. Results obtained with the unmodified all-oxygen compounds were compared with those obtained when phosphorothioate or alpha-DNA was used. In the wheat germ system a 17-mer sequence targeted to the coding region of beta-globin mRNA was specifically inhibitory when either the unmodified phosphodiester oligonucleotide or its phosphorothioate analogue were used. In contrast no effect was observed with the alpha-oligomer. These results were ascribed to the fact that phosphorothioate oligomers elicit an RNase-H activity comparable to the all-oxygen congeners, while alpha-DNA/mRNA hybrids were a poor substrate. Microinjected Xenopus oocytes followed a similar pattern. The phosphorothioate oligomer was more efficient to prevent translation than the unmodified 17-mer. Inhibition of beta-globin synthesis was observed in the nanomolar concentration range. This result can be ascribed to the nuclease resistance of phosphorothioates as compared to natural phosphodiester linkages, alpha-oligomers were devoid of any inhibitory effect up to 30 microM. Phosphorothioate oligodeoxyribonucleotides were shown to be non-specific inhibitors of protein translation, at concentrations in the micromolar range, in both cell-free systems and oocytes. Non-specific inhibition of translation was dependent on the length of the phosphorothioate oligomer. These non-specific effects were not observed with the unmodified or the alpha-oligonucleotides.

Vascular Smooth Muscle Cell Proliferation and Regrowth after Mechanical Injury in Vitro Are. Egr-1/NGFI-A-Dependent

“…Substitution of the phosphodiester backbone of native DNA with phosphorothioate moieties markedly augments resistance to nuclease digestion. 35,36 Phosphorothioates, however, have also been found to bind with high affinity to a large number of proteins 37,38 presumably the result of alterations in the presentation of negative charges. Without appropriate controls, sequence-independent effects caused by phosphorothioate chemistry can be mistakenly attributed to a true antisense effect.…”

Antisense oligonucleotides in solution or encapsulated in immunoliposomes inhibit replication of HIV-1 by several different mechanisms

“…Antisense oligonucleotides can block translation by an RNase-H-independent process if they are complementary to the 5' non coding region of the message or to the splice acceptor site (29,(31)(32)(33)(34). In contrast, if they are directed against the coding region the inhibition is RNase-H dependent.…”

Selective Anti-Gene Therapy for Cancer: Principles and Prospects.

“…Oligodeoxynucleotides and their analogs cause both selective and non-selective inhibition of expression in cell-free systems (Ghosh et al 1991a, b). Inhibition of /3-globin expression in Xenopus oocytes was chosen to compare the efficacy of different oligo analogs while avoiding the problems of relative cellular uptake, since the globin mRNA and the oligos are injected (Cazenave et al 1989). The amount of 35S-methionine incorporated into f3-globin expressed in the presence of an antisense oligo was compared with a control, and it was found that a natural (P0) anti-f3-globin-17-mer was quite effective in inhibiting expression, but that the S-oligo was even more effective.…”